PM_2.5 Exposure On Infectivity And Pro-inflammatory Effects Of Influenza Virus （H₃N₂） On Human Bronchial Epithelial Cells And Underlying Mechanism

Background PM_2.5（fine particu Late matter,also known as fine particu Late matter）pollution has become a prominent public health problem.Acute and chronic exposure to PM_2.5 can cause diseases of the respiratory system,cardiovascu Lar system,nervous system,reproductive system and other aspects.Respiratory epithelial cells are an important line of defense against inhaled foreign poisons,and are also the common target cells of PM_2.5 and influenza virus action.PM_2.5 and influenza viruses can cause their inflammatory response and modu Late the associated immune response.The main objective of this study was to explore the effect of PM_2.5 exposure on human bronchial epithelial cell lines（BEAS-2B）and induction of cell inflammation in H₃N₂virus infection.It further explores whether PM_2.5 affects H₃N₂virus infection with BEAS-2B cells through TLR4 and NF-κB pathways,which provides an important scientific basis for the development of intervention PM_2.5 to promote respiratory viral infection.Objective To investigate the effect and mechanism of PM_2.5 exposure on human respiratory epithelial cells（BEAS-2B）infected with H3N2 virus and inducing inflammatory response.Methods （1）Human respiratory epithelial cell line（BEAS-2B）:Normal human bronchial epithelial cells.Cell lines were initially infected by using adenovirus 12-SV40 hybrid virus infection and established by continuous cell passage.Cell cu Lture was performed using a single-layer adherent cu Lture method using DMEM complete medium containing 10%serum and 1%penicillin and streptomycin.（2）Determination of the effect of PM_2.5 and H₃N₂ virus exposure on the expression of inflammatory proteins and antiviral proteins in human bronchial epithelial cells:BEAS-2B cells were stimu Lated using PM_2.5suspensions with concentrations of 0,6.25,12.5 and 25μg/m L,respectively.Cell cu Lture was collected after 28 h and the concentrations of the cytokines interleukin-6（interleukin-6,IL-6）,interleukin-8（IL-8）and IFN-β（interferon-β,IFN-β）were detected by enzyme linked immunosorbent assay（ELISA）method Beas-2B cells were infected with different doses of H₃N₂ virus（MOI=0,MOI=1,MOI=2）,and the concentrations of IL-6,IL-8 and IFN-βin the cu Lture solution were determined by ELISA method 24 h later.（3）Determination of the effect of PM_2.5 on H₃N₂ virus infection with BEAS-2B cells:the experiment was divided into four groups(control group,PM_2.5 group,H₃N₂virus group,PM_2.5+H₃N₂ virus group),Using PM_2.5μg/m L PM_2.5 to stimu Late BEAS-2B cells for 4 h,adding H₃N₂virus（MOI=1）,and using immunoperoxidase monolayer after 24 h assay（IPMA）counted the number of virus-infected BEAS-2B cells and determined the concentrations of IL-6,IL-8 and IFN-βin the cu Lture medium supernatant by ELISA method.（4）Nuclear factor kappa-B（NF-κB）pathway mediates the effect of PM_2.5and H₃N₂virus on bronchial epithelial cells:the amount of IκBαprotein was determined after treating cells with PM_2.5 and H₃N₂ virus,and the amount of IκBαprotein in the PM_2.5 group,H₃N₂virus group,PM_2.5+H₃N₂ virus group was reduced compared with the control group,and the amount of IκBαprotein was reduced,and PM_2.5+The H₃N₂ virus group was significantly lower than the H₃N₂ virus group and the PM_2.5 group.This shows that PM_2.5 and H₃N₂both stimu Lated the activation of NF-κB,and the activation of NF-κB was significantly enhanced when exposed to PM_2.5 and H₃N₂,and the NF-k B pathway inhibitor Bay11-7085 pretreated BEAS-2B cells cou Ld significantly inhibit the effect of PM_2.5,H₃N₂virus or PM_2.5+H₃N₂on the expression of IL-6,IL-8 and IFN-βcells It also inhibits the cellu Lar infectivity of the virus increased by PM_2.5,and shows that NF-κB plays a key mediating role in the above-mentioned cellu Lar effects caused by PM_2.5 and H₃N₂ virus.（5）Effect of PM_2.5and H₃N₂virus on inflammatory transcription factor NF-κB:Cells were pretreated with NF-κB inhibitor Bay11-7085 for 1 h,and PM_2.5（12.5μg/m L）and H₃N2virus（MOI=1）were added,respectively.After 24 h of cu Lture,IPMA was used to count the number of VIRUS-infected BEAS-2B cells,and the concentrations of IL-6,IL-8 and IFN-βin the cell cu Lture liquid supernatant were detected by ELISA method.（6）To detect the role of TLR4 in PM_2.5 and H₃N₂ virus inducing BEAS-2B cell reactions:incubate with TLR4 neutralizing antibody and Ig G control antibody（2μg/m L）with BEAS-2B cells for 4 h,and then add PM_2.5（12.5μg/m L）or H₃N₂ virus（MOI=1）respectively.After24 hours,IPMA was used to count the number of BEAS-2B cells infected by the virus,and the concentrations of IL-6,IL-8 and IFN-βin the cell cu Lture solution were detected by ELISA method.Results （1）Effect of PM_2.5exposure on the expression of BEAS-2B cytokine:PM_2.5（0,6.25,12.5,25μg/m L）can increase the expression of inflammatory factors IL-6 and IL-8,inhibit the expression of the antiviral factor IFN-β,and the above effects show a concentration dependence.（2）Effect of H₃N₂ virus exposure on the expression of BEAS-2B cytokines:H₃N₂ virus（MOI=0,1,2）exposure can increase the expression of BEAS-2B cells IL-6,IL-8,IFN-β,and the above effects show a concentration dependence.（3）PM_2.5 exposure can increase the infectivity and inflammatory response of H₃N₂virus to BEAS-2B cells:pretreatment of BEAS-2B cells with PM_2.5 can significantly increase the number of cellu Lar infections and inflammatory effects of H₃N₂ virus,and reduce the cellu Lar antiviral effect.（4）Nuclear factor kappa-B（NF-κB）pathway mediates the effect of PM_2.5and H₃N₂virus on bronchial epithelial cells:the amount of IκBαprotein was determined after treating cells with PM_2.5 and H₃N₂ virus,and the amount of IκBαprotein in the PM_2.5 group,H₃N₂virus group,PM_2.5+H₃N₂ virus group was reduced compared with the control group,and the amount of IκBαprotein was reduced,and PM_2.5+The H₃N₂virus group was significantly lower than the H₃N₂ virus group and the PM_2.5group.This shows that PM_2.5 and H₃N₂both stimu Lated the activation of NF-κB,and the activation of NF-κB was significantly enhanced when exposed to PM_2.5and H₃N₂,and the NF-κB pathway inhibitor Bay11-7085 pretreated BEAS-2B cells cou Ld significantly inhibit the effect of PM_2.5,H₃N₂ virus or PM_2.5+H₃N₂ on the expression of IL-6,IL-8 and IFN-βcells It also inhibits the cellu Lar infectivity of the virus increased by PM_2.5,and shows that NF-κB plays a key mediating role in the above-mentioned cellu Lar effects caused by PM_2.5 and H₃N₂ virus.Conclusions PM_2.5 exposure can upregu Late the expression of BEAS-2B cell inflammatory proteins caused by H₃N₂ virus and downregu Late the expression of antiviral proteins,increase the cellu Lar appeal of virus,and both TLR4 and NF-κB signaling pathways are involved in regu Lating the expression of the above inflammatory proteins and antiviral proteins induced by PM_2.5and H₃N₂ virus.